4-pentylcyclohexanone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 22 - May 22, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 12, 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-pentylcyclohexanone

EC Number:

406-670-4

EC Name:

4-pentylcyclohexanone

Cas Number:

61203-83-6

Molecular formula:

C11H20O

IUPAC Name:

4-pentylcyclohexan-1-one

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Chbb: THOM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Thomae GmbH, Biberach, Germany
- Age at study initiation: aged 9 weeks
- Weight at study initiation: mean weight of males 302g (287-319 g) and females 196 g (184-208 g)
- Housing: individually in type III Makrolon cages
- Diet: ad libitum, Altromin Standard Diet TPF® N 1324
- Water: ad libitum, tap water

DETAILS OF FOOD AND WATER QUALITY: The diet was checked periodically by an independent and German Government approved testing laboratory, according to the regulations of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. Drinking water employed in this type of study was regularly investigated microbiologically, physico-chemically, and chemically.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-28
- Humidity (%): 26-45
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Methocel® K4M Premium/Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle. Every week three test material concentrations were prepared and stored in brown glass bottles. Before application the test material suspensions were stirred intensively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

After storing for 1 week in 0.5 % aqueous hydroxypropyl-methylcellulose Tween 80 (5 g Methocel® K4M Premium + 5 g Tween 80), in brown glass bottles under protection from light, no alteration in the concentration of the test material was found. There was a moderate decrease in concentrations with time, in the lower concentrations mainly, after the 6th day. Therefore after the 7th day fresh preparations were used.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, 7 times per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 1000 mg/kg are recommended as highest dose for a 4-week-toxicity-study in the OECD-guidelines. The lower doses were always half of the higher dose.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
once weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in treatment week 4
- Anaesthetic used for blood collection: Yes, halothane anesthesia
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: Erythrocyte count (ERY), Leukocyte count (LEUK), Hemoglobin (HB), Packed cell volume (PCV), Platelet count (PLAT), Reticulocyte count (RET), Differential blood cell count % Absolute number of segmented neutrophilic granulocytes(SE), Absolute number of lymphocytes (LY), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin pg content (MCH), Mean corpuscular hemoglobin g/dl concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in treatment week 4
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus, Glucose, Urea, Creatinine, Bilirubin Cholesterol, Triglycerides, Alanine aminotransferase, Alkaline phosphatase Aspartate aminotransferase, Protein (total), Albumin

URINALYSIS: Yes
- Time schedule for collection of urine: In study week 4,
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, animals without feed for 18 hours
- Parameters checked: pH value, Protein, Glucose, Urobilinogen, Bilirubin, Blood, Sediment

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to start of treatment and in study weeks 1 and 4 ophthalmologic examinations were performed in all rats. The anterior chamber of the eye was examined directly with an ophthalmoloscope (Carl Zeiss, No. 29902). The fundus of the eye was examined (indirectly) with a hand slit lamp and a lens

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The organs below were fixed in ca. 10% formaldehyde solution. In addition a liver sample from the lobus sinister lateralis of the surviving rats was fixed in Lison-Vokaer glycogen fixative. Then the organs for which a stain is given were embedded in paraffin, and sections of them were stained as follows:
Heart, Aorta thoracalis, Lung, Liver, Spleen, Kidneys, Testes, Epididymides, Prostate, Seminal vesicles, Uterus, Ovaries, Urinary bladder, Stomach, Pars proventricularis, Pars fundica, Pars pylorica, Small intestine, Duodenum, Jejunum, Ileum
Large intestine, Caecum, Colon ascendens, Rectum, Mesenterial vessels, Adrenals, Lymph nodes, Lnn. maxillares ant., Lnn. mesenteriales, Thyroids, Parathyroids, Thymus, Pancreas, Bone, Bone marrow (femur), Trachea, Esophagus, Salivary gland Diaphragm, Thigh muscle, Sciatic nerve, Cerebrum, Cerebellum, Medulla oblongata, Spinal cord (I.lumbalis), Pituitary, Adipose tissue (retroperitoneal), Skin with mammary glands, R. thoracalis dextra and/or R. inguinalis sinistra, Eyes, Tongue, Larynx, Nasal and ethmoidal mucosa Middle ear, inner ear

HISTOPATHOLOGY: Yes

The following weights were determined for the surviving rats which were sacrificed at the scheduled times: Body weight (after exsanguination)
Heart, Liver, Kidneys (together), Spleen, Thymus, Testes or ovaries (together) Prostate or uterus, Adrenals (together), Thyroid glands with parathyroids (together), Pituitary, Brain, Eyes (together)
After removing of the ossa frontalia, the skull with brain, pituitary, and eyes, was fixed in ca. 10 % formaldehyde solution for about 2 weeks. The thyroids were left at the larynx and fixed for about 2 weeks in ca. 10% formaldehyde, also. After fixation brain, pituitary, eyes, and thyroids were dissected and weighed. The relative organ weights were calculated per 100 g body weight.

Statistics:

Body weight gain, food consumption, water consumption, organ weights, the clinico-chemical serum parameters and the haematological parameters haematocrit, haemoglobin and numbers of erythrocytes and leucocytes of the dose groups were compared with those of the control (separately for each sex and time) using the multiple two-sided t-test according to DUNNETT (1955).
Different group sizes or variance non- homogeneity between the dose groups and the control were considered by correcting the critical t-values according to DUNNETT (1964), and in case of variance non-homogeneity by WELCH'S correction of the degrees of freedom (cf. WINER, 1970, pp. 36 - 39).
In cases of very low group sizes or extreme variance non-homogeneity no statistical comparison was performed.
For evaluation of the haematology parameters reticulocytes, eosinophilic, basophilic, juvenile/ bandform neutrophilic granulocytes and the leucocytes (which were not classified further) the procedure of STUCKY and VOLLMAR (1976) (which is based on a linear rank test acc. to KRAUTH, 1971) with the two sided alternative was used.
The parameters segmented neutrophilic granulocytes (SE) and lymphocytes (LY) were evaluated together acc. to UNKELBACH (1980), using the same procedure for the values SE/(SE +LY) .

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

From the lowest dose of 250 mg/kg onwards, all rats showed salivation and burrowing in the litter, symptoms which are often seen after administration of substances with bad taste. Abdominal, and at times lateral, position was seen sporadically in rats of group 3 (500 mg/kg) and frequently in group 4 (1000 mg/kg). The symptom was followed by reduced spontaneous activity up to about 3 hours after treatment. In the course of the study the symptoms disappeared in group 3, but were observed in group 4 up to sacrifice. Chromodakryorrhea was seen once (rat No. 19, group 4). Rat No. 54 (group 4 f) showed a blood-crusted nose and bad general condition on study day 9. Therefore treatment of this animal was omitted on day 9, and then continued on day 10.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two rats died in the course of the study. Rat No. 9 m (group 2, 250 mg/kg) showed dyspnea directly after treatment for some hours and was therefore sacrificed. The necropsy findings indicated false application.
Rat No. 52 f (group 4, 1000 mg/kg) was found dead on study day 8. The clinical symptoms indicate toxic effects of the test material as cause of death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences in body weight gain from the controls were seen in male or female rats of the 250 mg/kg and 500 mg/kg group after the 4 week treatment. The males from the 1000 mg/kg group showed reduced body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in the males of the 1000 mg/kg group

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption in the 250 and 500 mg/kg groups corresponded to that of the controls, but was increased in both sexes of the 1000 mg/kg group.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

As a finding, which is characteristic for young rats of this rat strain, rests of the membrana pupillaris persis-tens were seen in treated and untreated rats with the same frequency.

Haematological findings:

no effects observed

Description (incidence and severity):

There was no indication of effects of the test materials on the red or white blood cell counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In all test material treated rats a dose-dependent increase of serum cholesterol values was seen. The values were significantly different from the controls in the males of groups 3 and 4 and in the females of group 4. In group 4 increased triglyceride values were seen in addition. In males a dose-dependent increase in total serum protein values was observed, which, however, was only significant in the highest dose group. The alkaline phosphatase was also significantly increased in this group. The females of group 4 showed a slight increase in ALAT activity.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The decreased thymus weights and the Increased adrenal weights, seen after the highest dose (1000 mg/kg), had no histological correlate and were considered as adaptation syndrome. The increased liver weight (also seen only in the highest dose group) is a consequence of increased metabolic activity. The only finding was reduced glycogen content.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic finding of thickening of the stomach wall, seen in the 1000 mg/kg group, was diagnosed as inflammation with hyperkeratosis, and was seen histologically from 500 mg/kg on.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: In rats of the highest dose group the only finding different from the controls was reduced glycogen contents, an unspecific finding which has to be expected as consequence of increased metabolic activity. Thus, histopathologic examination of the livers from the rats of the lower dose groups was considered unnecessary.
Stomach: In the forestomach test material induced dose-dependent acute inflammations and consequent hyperkeratosis were found.
Rat No. 9, which was sacrificed after false application, showed necrotizing tracheitis and hemorrhages in the lungs. In rat No. 52, which died, dilatation of the right ventricle of the heart, lung edema, hydropic liver cell degeneration, and liver hemorrhages, which are signs of shock, were found.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL after oral application was determined to be 250 mg/kg bw/day for males and females after 28 days administration to males and female rats.

Executive summary:

A study according OECD TG 407 was conducted to determine the oral toxicity after repeated administration of the test item to Wistar rats by gavage. The test material was administered as suspension in aqueous 0.5 % Methocel® K4M Premium/Tween 80.

Both substances were given to rats in daily oral doses of 250, 500, and 1000 mg/kg bw/day. Each group consisted of 10 animals (5 m, 5 f).

Appearance and behaviour of all rats were checked daily. Body weight and water consumption were determined twice weekly and food consumption once weekly. In weeks 1 and 4 ophthalmological inspections were performed.

Haematological examinations and clinico-chemical analyses of serum and urine were carried out in all animals in treatment week 4. At the end of the 4-week treatment period all animals were sacrificed and necropsied.

12 organs were weighed from each animal and a maximum of 38 organs and tissues per animal were subjected to histopathological examination.

One rat of the 1000 mg/kg group died due to shock as consequence of treatment.

The histological findings were dilatation of the right side of the heart, lung edema, hydropic liver cell degeneration, and haemorrhages in the liver.

Due to the stinging smell of the test material suspension salivation and burrowing in the litter were seen in all treated rats directly after administration.

In the 500 mg/kg bw/day group, and more pronounced in the 1000 mg/kg bw/day group, abdominal and lateral position were observed after treatment, which were followed by reduced spontaneous activity for several hours.

The males from the 1000 mg/kg bw/day group showed reduced body weight gain and food consumption, but water consumption was increased in both sexes in this group.

The haematological examinations gave no indication of test material effects on the blood picture.

The clinico-chemical examinations revealed a dose-dependent increase in serum cholesterol values, which was pronounced from 500 mg/kg bw/day onwards. In the 1000 mg/kg bw/day group increased triglyceride values were seen also, and in males only, increased total protein values in addition. The activity of the alkaline phosphatase was increased and that of the alanine-aminotransferase, in females only, slightly increased in this group.

There were no ophthalmological findings related to the test substance. The macroscopic finding of thickening of the stomach wall, seen in the 1000 mg/kg bw/day group, was diagnosed as inflammation with hyperkeratosis, and was seen histologically from 500 mg/kg bw/day on. The decreased thymus weights and the increased adrenal weights, seen after the highest dose (1000 mg/kg bw/day), had no histological correlate and were considered as adaptation syndrome. The increased liver weight (also seen only in the highest dose group) is a consequence of increased metabolic activity. The only finding was reduced glycogen content.

The NOAEL after oral application was determined to be 250 mg/kg bw/day for males and females after 28 days administration to males and female rats.